PRETREATMENT METHOD, PRESERVATION METHOD, AUTOMATIC TREATMENT SYSTEM AND DETECTION METHOD FOR URINE SAMPLE

Abstract

The invention discloses a pretreatment method, a preservation method, an automatic treatment system and a detection method for a urine sample, and directs to the technical field of biological detection. The pretreatment method comprises subjecting a urine sample after protein lysis to a reductive alkylation treatment, followed by protein enrichment and enzymolysis. The protein enrichment is performed on the sample after the reductive alkylation treatment using a PVDF filter plate for protein enrichment; The invention also provides an automatic treatment system and an automatic sample treatment method. The treatment system greatly reduces the labor intensity of people, is beneficial to facilitate the treatment efficiency of urine sample treatment, meets the requirements of high-flux and automated pretreatment of the proteomics, and accommodates the reproducibility and flux of current clinical needs.

Claims

1. A preservation method for a urine sample, comprising: subjecting the urine sample after protein lysis to a reductive alkylation treatment, and then followed by a protein enrichment; wherein the protein enrichment is performed on the sample after the reductive alkylation treatment using a PVDF filter plate for protein enrichment; the mixture volume ratio of a lysate used for protein lysis to the urine sample to be lysed is 1:0.1-9.

2. The preservation method for the urine sample according to claim 1, characterized in that the lysate is at least one selected from the group consisting of urea, thiourea, guanidine hydrochloride, tris (hydroxymethyl) aminomethane-hydrochloride, phenylmethylsulfonyl fluoride, sodium dodecyl sulfate, sodium deoxycholate and 3-[3-(cholamidopropyl) dimethylammonio]-1-propanesulfonate; preferably, the lysate is selected from urea and the final concentration of the urea in the urine sample to be lysed is 1M-5M; preferably, a diluent for the protein lysate is at least one selected from the group consisting of ammonium bicarbonate, tris (hydroxymethyl) aminomethane-hydrochloride solution, phosphate solution.

3. The preservation method for the urine sample according to claim 1, further comprising, before the protein enrichment, activating the PVDF filter plate, equilibrating with the lysate after the activation, and thereafter transferring the sample after the reductive alkylation treatment to the equilibrated PVDF filter plate for protein enrichment; preferably, an activating agent for the activation is an alcohol.

4. A pretreatment method for a urine sample, comprising: subjecting the urine sample after protein lysis to a reductive alkylation treatment, then protein enrichment, thereafter enzymolysis, and concentrating and lyophilizing; wherein the protein enrichment is performed on the sample after the reductive alkylation treatment using a PVDF filter plate for protein enrichment; the mixture volume ratio of a lysate used for protein lysis to the urine sample to be lysed is 1:0.1:9.

5. The pretreatment method for the urine sample according to claim 4, characterized in that it also comprises collecting a filtrate from the PVDF filter plate after the enzymolysis; preferably, the enzymes for the enzymolysis are trypsin and lysinase; preferably, the time for enzymolysis is 1-18 h; preferably, the protein enrichment comprises adding the sample after the reductive alkylation treatment to the PVDF filter plate, and after centrifugation, washing the centrifuged sample with an eluent.

6. The pretreatment method for the urine sample according to claim 4, characterized in that the lysate is at least one selected from the group consisting of urea, thiourea, guanidine hydrochloride, tris (hydroxymethyl) aminomethane-hydrochloride, phenylmethylsulfonyl fluoride, sodium dodecyl sulfate, sodium deoxycholate and 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate; preferably, the lysate is selected from urea and the final concentration of the urea in the urine sample to be lysed is 1M-5M.

7. An automatic treatment system for a urine sample, comprising a urine sample storage unit, a treating fluid supply unit, a PVDF filter plate supply unit, a sample suction unit, a protein collection unit and an enzyme storage unit, wherein the urine sample storage unit, the treating fluid supply unit, the PVDF filter plate supply unit, the sample suction unit, the protein collection unit and the enzyme storage unit are electrically connected to a control terminal for automatic control.

8. The automatic treatment system for the urine sample according to claim 7, characterized in that the automatic treatment system further comprises a lysis reaction vessel supply unit, a shaker, a concentrator, and a PCR plate; wherein the treating fluid supply unit includes a lysate supply unit, a reducing agent supply unit, an alkylating agent supply unit, an alkylation reaction terminating agent supply unit, an activating agent supply unit, an eluent supply unit, and a reconstitution solvent supply unit.

9. A treatment method for a urine sample by using the automatic treatment system for the urine sample according to claim 7, comprising: (1) protein lysis: taking a urine sample to be tested from a urine sample storage unit into a lysis reaction vessel by using a sample suction unit, and sucking a lysate from a treating fluid supply unit into the lysis reaction vessel via the sample suction unit to perform the protein lysis; (2) reductive alkylation: sucking a reducing agent from the treating fluid supply unit into the lysis reaction vessel via the sample suction unit to perform a reduction reaction, sucking an alkylating agent from the treating fluid supply unit into the lysis reaction vessel via the sample suction unit to perform an alkylation reaction, and then sucking an alkylation reaction terminating agent from the treating fluid supply unit via the sample suction unit to terminate the alkylation reaction; (3) protein enrichment: activating the PVDF filter plate by sucking an activating agent from the treating fluid supply unit into the PVDF filter plate via the sample suction unit, equilibrating the PVDF filter plate by sucking the lysate from the treating fluid supply unit into the PVDF filter plate via the sample suction unit, then adding a product after the reductive alkylation treatment to the PVDF filter plate via the sample suction unit, and centrifuging; (4) proteolysis: sucking an enzyme reaction solution from an enzyme storage unit into the PVDF filter plate via the sample suction unit to perform an enzymolysis reaction, then sucking an eluent from the treating fluid supply unit into the PVDF filter plate via the sample suction unit to elute an enzymolysis reaction product, and then combining the eluent; and (5) concentrating and lyophilizing: concentrating and lyophilizing the eluent.

10. A method of mass spectrometric detection for a urine sample, which is directed for the purpose of non-diagnosis of disease, characterized by comprising: pre-treating the urine sample by using the method of claim 9, and then performing peptide fragment detection by using a mass spectrometer; setting a mobile phase A as an aqueous solution containing 0.05-0.2% formic acid and a mobile phase B as 80% acetonitrile containing 0.05-0.2% formic acid for gradient elution, with a flow rate of 200-300 nl/min and a column temperature of 30-55° C.; preferably, the gradient elution has procedures of 1-6 min, 1%-8% B, 6-30 min, 8-99% B; setting mass spectrometry parameters, including a mass spectrum full scan resolution of 240,000, 120,000, 70,000, 60,000, 45,000, 30,000, 17,500, 15,000 or 7,500@m/z 200, AGC of 1E5-3E6, maximum ion sample injection time of 10-100 ms, a scan range of m/z 200-2000, normalized collision energy of 15-27%; a secondary mass spectrum scan resolution of 240,000, 120,000, 70,000, 60,000, 45,000, 30,000, 17,500, 15,000 or 7,500@m/z 200, a scanning range of m/z 200-2000, an AGC of 1E5-1E6, maximum ion injection time of 10-100 ms, dynamic exclusion time of 10-40 s, and a charge valence state of 2.sup.+-8.sup.+.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0061] In order to more clearly describe the technical solutions in the embodiments of the invention, the drawings to be used in the embodiments will be briefly introduced below. The drawings in the following description are only some embodiments of the invention, and thus should not be deemed as limiting the scope of the invention. It will be apparent to those skilled in the art that other drawings may be obtained from the drawings without any creative efforts.

[0062] FIG. 1 is a flow chart of urine sample preservation and pretreatment;

[0063] FIG. 2 is a software operation interface for urine sample preservation and automated pretreatment of the proteomics;

[0064] FIG. 3 shows an internal structure of a workstation for urine sample preservation and automated pretreatment of the proteomics (1. thermostatic mixing shaker; 2. 200 μL pipette tip; 3. PCR plate; 4. 50 μL pipette tip; 5-PCR plate; 6-low temperature disk; 7. twelve-channel tank; 8. 0.5 mL 96-well plate; 9. PVDF filter plate);

[0065] FIG. 4 is a ranking graph of protein distribution of all samples;

[0066] FIG. 5 is a statistical graph of the results of protein identification of samples;

[0067] FIG. 6 is a statistical graph of identification of protein and peptide fragments thereof corresponding to different concentrations of urea, with black representing the number of proteins identified and gray representing the number of peptide fragments identified;

[0068] FIG. 7 is a statistical graph of identification of proteins and peptide fragments thereof corresponding to different preservation times, with black representing the number of proteins identified and gray representing the number of peptide fragments identified.

DESCRIPTION OF THE EMBODIMENTS

[0069] In order to make the objectives, technical solutions, and advantages of the embodiments of the invention more apparent, the technical solutions in the embodiments of the invention will be described in detail in conjunction with the accompanying drawings in the embodiments of the present application. Where specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions suggested by the manufacturer. Where the reagents or instruments used are not specified by the manufacturer, they are conventional products commercially available.

[0070] The characteristics and performance of the invention are further described in detail in the following embodiments.

Comparative Example 1

[0071] A pretreatment method (shown with reference to FIGS. 1 and 2) for urine samples (A, B, C, D urine samples all from healthy volunteers) includes the following steps.

(1) Protein Lysis

[0072] Sample A: 100 μL of the same urine sample was added with 300 μL of 8M urea (diluent: 50 mM ammonium bicarbonate), with a final concentration of urea of 6M. The mixture was vortexed homogeneously to extract the protein.

(2) Reductive Alkylation

[0073] Dithiothreitol was added to a product after protein lysis to a final concentration of 10 mM, and the reaction thereof was carried out at room temperature for 20 min. Iodoacetamide (alkylation) was added to the reduced product to a final concentration of 20 mM, and the reaction thereof was carried out in the dark for 20 min. An equal volume of dithiothreitol was added to the alkylated product to neutralize the excess iodoacetamide in the alkylation reaction.

(3) Protein Enrichment

[0074] A PVDF filter plate activation was carried out by adding 200 μL 70% ethanol to the PVDF filter plate and centrifuging at 1000 g. PVDF filter plate equilibration was carried out by adding 200 μl of 6M urea (diluent: 50 Mm ammonium bicarbonate) to the PVDF filter plate, and centrifuging at 1000 g. The sample was then transferred to the PVDF filter plate and centrifuged at 1000 g. The sample was finally washed by adding 50 mM ammonium bicarbonate solution and centrifuged at 1000 g.

(4) Protein Enzymolysis

[0075] 100 μL of 50 mM ammonium bicarbonate solution and 1 μg of mixed trypsin and lysinase (LysC) were added. The mixture was shaken and incubated at 37° C. for 2 h, and centrifuged at 1000 g for 1 min after the completion of incubation to collect a filtrate. An additional 150 μL of 40% acetonitrile (containing 0.1% formic acid) was added to elute the peptide fragments and the eluent was combined.

(5) Concentrating and Lyophilizing

[0076] The collected eluent was concentrated and lyophilized in a vacuum centrifugal concentrator.

Comparative Example 2

[0077] The urea process is combined with a conventional pretreatment process as follows.

[0078] D samples were treated as follows. A 300 μL urine sample was added to 1500 μL of pre-cooled methanol according to the ratio of urine: methanol=1:5 (V/V). The mixture was vortexed for 20 s, allowed to stand at −20° C. for 1.5 h, and centrifuged for 10 min at 12000 g at 4° C. to discard a supernatant. The precipitate was washed once with 80% ethanol and dried in a concentrator. The sample was reconstituted with 50 μL of urea solution using the BSA method (Pierce™ BCA Protein Assay Kit, Brand: Thermo Fisher, Code: 23227) to determine the protein concentration. According to the protein concentration determined by the BSA method, 10 μg protein was added into a 96-well plate, and the mixture was made up to 50 μL of total volume by using 8 M urea. Dithiothreitol was added to a final concentration of 10 mM, and the reaction thereof was carried out at room temperature for 20 min. Iodoacetamide was added to a final concentration of 20 mM, and the reaction thereof was carried out for 20 min in the dark. An equal amount of dithiothreitol was added to neutralize the excess iodoacetamide. 1 μg of mixed trypsin and lysinase (LysC) was added. The mixture was incubated at 37° C. with shaking for 2 h, and 150 μL of 50 mM ammonium bicarbonate was added to dilute urea to below 2 M after the completion of incubation. The reaction was terminated by adding 20 μL of 10% trifluoroacetic acid to the reaction system, and followed by a desalting operation.

[0079] The desalting operation was as follows. 100 μL of methanol was added to the desalted plate and the mixture was centrifuged at 600 g for 1 min. 100 μL of 0.2% trifluoroacetic acid/80% acetonitrile was added and the mixture was centrifuged at 600 g for 1 min. 200 μL of 0.2% trifluoroacetic acid/water was added and the resultant was centrifuged at 600 g for 1 min. The sample was added and the mixture was centrifuged at 600 g for 1 min, which is repeated once. 200 μL of 0.2% trifluoroacetic acid/water was added and the mixture was centrifuged at 600 g for 1 min and rinsed. 100 μL of 0.2% trifluoroacetic acid/80% acetonitrile was added and the mixture was centrifuged at 600 g for 1 min for eluting. The filtrate was collected for concentration and lyophilization.

Embodiment 1

[0080] A pretreatment method for a urine sample is substantially the same as Comparative Example 1, except for the different concentration of the protein lysate as follows.

[0081] Sample B: 200 μL of the same urine sample was added with 200 μL of 8M urea (diluent: 50 Mm ammonium bicarbonate), with the final concentration of urea of 4M. The mixture was vortexed homogeneously to extract the protein.

Embodiment 2

[0082] A pretreatment method for a urine sample is substantially the same as Comparative Example 1, except for the different concentration of the protein lysate as follows.

[0083] Sample C: 300 μL of the same urine sample was added with 200 μL of 8M urea (diluent: 50 Mm ammonium bicarbonate), with the final concentration of urea of 3M. The mixture was vortexed homogeneously to extract the protein.

Embodiment 3

[0084] A preservation method for a urine sample (shown with reference to FIG. 1) includes the following steps.

(1) Protein Lysis

[0085] 300 μL of the same urine sample (sample C) was taken (diluent: 50 Mm ammonium bicarbonate), with the final concentration of urea of 3 M. The mixture was vortexed homogeneously to extract the protein.

(2) Reductive Alkylation

[0086] Dithiothreitol was added to a product after protein lysis to a final concentration of 10 mM, and the reaction thereof was carried out at room temperature for 20 min. Iodoacetamide (alkylation) was added to the reduced product to a final concentration of 20 mM, and the reaction thereof was carried out in the dark for 20 min. An equal volume of dithiothreitol was added to the alkylated product to neutralize the excess iodoacetamide in the alkylation reaction.

(3) Protein Enrichment

[0087] A PVDF filter plate activation was carried out by adding 200 μL 70% ethanol to the PVDF filter plate and centrifuging at 1000 g. PVDF filter plate equilibration was carried out by adding 200 μl of 3M urea (diluent: 50 Mm ammonium bicarbonate) to the PVDF filter plate, and centrifuging at 1000 g. The sample was then transferred to the PVDF filter plate and centrifuged at 1000 g. The sample was finally washed by adding 50 mM ammonium bicarbonate solution and centrifuged at 1000 g for 1 min.

[0088] Urine protein samples stored in the PVDF filter plate were stored at −80° C. for 1 month.

Embodiment 4

[0089] This embodiment provides a preservation method for an urine sample (as shown in FIG. 1), which differs from Embodiment 3 only in respect of the preservation period. In the embodiment, the urine protein samples stored in the PVDF filter plate were stored at −80° C. for 3 months.

Embodiment 5

[0090] This embodiment provides a preservation method for a urine sample (as shown in FIG. 1), which differs from Embodiment 3 only in respect of the preservation period. In the embodiment, the urine protein samples stored in the PVDF filter plate were stored at −80° C. for 5 months.

Embodiment 6

[0091] This embodiment provides a preservation method for a urine sample (as shown in FIG. 1), which differs from Embodiment 3 only in respect of the preservation period. In the embodiment, the urine protein samples stored in the PVDF filter plate were stored at −80° C. for 7 months.

Embodiment 7

[0092] This embodiment provides a preservation method for a urine sample (as shown in FIG. 1), which differs from Embodiment 3 only in respect of the preservation period. In the embodiment, the urine protein samples stored in the PVDF filter plate were stored at −80° C. for 9 months.

Embodiment 8

[0093] This embodiment provides a preservation method for a urine sample (as shown in FIG. 1), which differs from Embodiment 3 only in respect of the preservation period. In the embodiment, the urine protein samples stored in the PVDF filter plate were stored at −80° C. for 12 months.

Embodiment 9

[0094] This embodiment provides an automatic treatment system for a urine sample. Specifically, the proteome pretreatment process of clinical urine samples is integrated into an automatic workstation. With reference to FIG. 3, the internal structure of the workstation includes a thermostatic mixing shaker 1, a 200 μL pipette tip 2, a PCR plate 3, a 50 μL pipette tip 4, a PCR plate 5, a low temperature disk 6, twelve-channel tank 7, a 0.5 mL 96-well plate 8 and a PVDF filter plate 9. The entire automation process can be divided into five parts: protein lysis, reductive alkylation, protein enrichment, protein digestion and concentration and lyophilization.

[0095] The automatic treatment system includes a urine sample storage unit, a treating fluid supply unit, a PVDF filter plate supply unit, a sample suction unit, a protein collection unit and an enzyme storage unit, wherein the urine sample storage unit, the treating fluid supply unit. The PVDF filter plate supply unit, the sample suction unit, the protein collection unit and the enzyme storage unit are electrically connected to a control terminal for automatic control.

[0096] The above treatment system further includes a lysis reaction vessel supply unit, a shaker, a concentrator and a PCR plate.

[0097] The treating fluid supply unit includes a lysate supply unit, a reducing agent supply unit, an alkylating agent supply unit, an alkylation reaction terminating agent supply unit, an eluent supply unit, an activating agent supply unit, and a reconstitution solvent supply unit. The treating fluid supply unit may be a twelve-channel tank, with a different reagent supply unit provided in each channel.

[0098] In this embodiment, the control terminal is a computer. The functions of automatic liquid supply, elution, sample loading, shaking and enrichment are realized by the control terminal.

[0099] Specifically, the automatic treatment system performs an automated urine sample treatment process as follows, and a more specific pretreatment experimental process is shown in Table 1.

[0100] Step 1-Protein lysis (as in Embodiment 2): A 300 μL of urine sample was transferred automatically and placed in a 0.5 mL 96-well plate and on a thermostatic mixing shaker at Position 1. Then, 200 μL of 8M urea (diluent: 50 Mm ammonium bicarbonate) was sucked and added into the 0.5 mL 96-well plate on the thermostatic mixing shaker at Position 1, respectively. The mixture was vortexed at a rotation speed of 1000 rpm to extract protein.

[0101] Step 2-reductive alkylation: 10 μL of 0.5 M dithiothreitol was sucked from Column 2 (A2) of twelve-channel tank at Position 7 and added into 0.5 mL 96-well plate on the thermostatic mixing shaker at Position 1 respectively for a final concentration of 10 mM. The mixture was vortexed homogeneously at the rotation speed of 1000 rpm, and reacted at room temperature for 20 min. 20 μl of 0.5 M iodoacetamide was sucked from Column 3 (A3) of the twelve-channel tank at Position 7 and added into the 0.5 mL 96-well plate on the thermostatic mixing shaker at Position 1 for a final concentration of 20 mM. The mixture was vortexed homogeneously at the rotation speed of 1000 rpm, and reacted in the dark for 20 min. Then, 10 μL of 0.5 M dithiothreitol was sucked from Column 2 (A2) of the twelve-channel tank at Position 7 and added into the 0.5 mL 96-well plate on the thermostatic mixing shaker at Position 1, respectively. The mixture was vortexed homogeneously at the rotation speed of 1000 rpm to neutralize excess iodoacetamide.

[0102] Step 3-Protein Enrichment: 200 μL of 70% ethanol was sucked from Column 4 (A4) of the twelve-channel tank at Position 7 and added into the PVDF-96 well plate (namely, a PVDF filter plate) at Position 9 respectively. The mixture was centrifuged at 1000 g to activate the PVDF filter plate. 200 μL of 3 M urea (diluent: 50 Mm ammonium bicarbonate) was sucked from Column 4 (A5) of the twelve-channel tank at Position 7 added into the PVDF filter plate at Position 9, respectively. The mixture was centrifuged at 1000 g for PVDF filter plate equilibration. Then, the sample after the completion of reductive alkylation in the 0.5 mL 96-well plate on the thermostatic mixing shaker at Position 1 was transferred into PVDF filter plate at Position 9, and the same was centrifuged at 1000 g. Finally, 100 μL of 50 mM ammonium bicarbonate solution was sucked from Column 6 (A6) of the twelve-channel tank at Position 7 to wash the sample, and the mixture was centrifuged at 1000 g.

[0103] Step 4-Protein digestion: 100 μL of 50 mM ammonium bicarbonate solution and 1 μg of mixed trypsin and lysinase (LysC) were sucked from Column 1 of the low-temperature disk at Position 6 (that is the enzyme storage unit) and respectively added into the PVDF filter plate at Position 9. Then, the PVDF filter plate at Position 9 was displaced to the thermostatic mixing shaker at Position 1 for shaking incubation at 37° C. at a rotation speed of 1000 rpm for 2 h. After the incubation is completed, the mixture was centrifugated at 1000 g for 1 min for collecting a peptide fragment filtrate. 150 μL of 40% acetonitrile (containing 0.1% formic acid) solvent was sucked from Column 7 (A7) of the twelve-channel tank at Position 7, and added into PVDF filter plate at Position 9 for elution. The mixture was centrifugated for 1 min at 1000 g, and all the eluents were combined.

[0104] Step 5-Concentrating and lyophilizing: the collected eluent was concentrated and lyophilized in a vacuum centrifugal concentrator.

[0105] According to the requirements of mass spectrometry detection, the automatic treatment system of the present application can be further used for a reconstitution operation. The concentrated and lyophilized peptide fragment sample was placed on the thermostatic mixing shaker at Position 1 of the workstation. 20 μL of 0.1% formic acid aqueous solvent was sucked from Column 8 (A8) of the twelve-channel tank at Position 7. The mixture was vortexed homogeneously at a rotation speed of 1000 rpm for 1 min to perform peptide reconstitution. After the completion of reconstitution, 15 μL of the supernatant was transferred from the 0.5 mL 96-well plate on the thermostatic mixing shaker at Position 1 into the PCR plate at Position 3, respectively, waiting for mass spectrometry detection and analysis to perform peptide fragment detection.

TABLE-US-00001 TABLE 1 Automatic treatment process Pretreatment experimental Time process Automatic operation Disk position Reagent consuming Protein 1. A robotic arm 1. A 0.5 ml 96-well 1. Urine 1. 30 min lysis removed a 300 μL of plate sample disk was sample 2. 1 min urine sample and placed on the 2. 8M urea 3. 1 min placed the same in a thermostatic mixing solvent 0.5 mL 96-well plate. shaker at Position 1. 2. The robotic arm 2. 8M Urea solvent removed 200 μL of was placed in Column 8M urea (diluent: 50 1 (A1) of twelve- mM ammonium channel tank (which is bicarbonate) and the treating fluid added the same to supply unit) at Position the 0.5 mL 96-well 7. plate in the previous step. 3. The mixture was vortexed homogeneously at the rotation speed of 1000 rpm for extract of the protein. Reductive 1. 10 μL of 0.5M 1.1 The 0.5 mL 96-well 1. 0.5M 1.1 1 min alkylation dithiothreitol was plate sample disk was dithiothreitol 1.2 20 min transferred and placed on the 2. 0.5M 2.1 1 min added into the 0.5 mL thermostatic mixing iodoacetamide 2.2 20 min 96-well plate in the shaker at Position 1. 3. 0.5M 3. 1 min previous step by the 1.2 0.5M dithiothreitol dithiothreitol robotic arm. The was placed in Column mixture was vortexed 2 (A2) of the twelve- homogeneously at channel tank (namely, the rotation speed of the treating fluid 1000 rpm, and supply unit) at Position reacted at room 7; temperature for 20 2. 0.5M min; iodoacetamide was 2. 20 μL of 0.5M placed in Column 3 iodoacetamide was (A3) of the twelve- transferred into the channel tank (which is 0.5 mL 96-well plate the treating fluid in the previous step supply unit) at Position by robotic arm. The 7. mixture was vortexed 3. 0.5M dithiothreitol homogeneously at was placed in Column the rotation speed of 2 (A2) of the twelve- 1000 rpm, and channel tank (which is reacted at room the treating fluid temperature in the supply unit) at Position dark for 20 min; 7. 3. 10 μL of 0.5M dithiothreitol was transferred and added into the 0.5 mL 96-well plate in the previous step with the robotic arm. The mixture was vortexed homogeneously at the rotation speed of 1000 rpm. Protein 1. The robotic arm 1.1 70% ethanol was 1.1 70% 1. 1 min enrichment transferred 200 μL of placed in Column 4 Ethanol 2. 1 min 70% ethanol and (A4) of the twelve- 2. 3M Urea 3. 1 min respectively added channel tank at (diluent: 50 4. 1 min the same into a Position 7. mM PVDF-96 well plate 1.2 PVDF filter plates ammonium (namely, a PVDF were placed at bicarbonate) filter plate) to activate Position 9. 4. 50 mM the PVDF filter plate. 2. 3M Urea (diluent: 50 ammonium 2. The robotic arm Mm ammonium bicarbonate transferred 200 μL of bicarbonate) was solution 3M urea (diluent: 50 placed in Column 4 mM ammonium (A5) of the twelve- bicarbonate) and channel tank at respectively added Position 7. the same into the 3. The 0.5 mL 96-well PVDF filter plate plate sample disk was respectively for placed on the PVDF filter plate thermostatic mixing equilibration. shaker at Position 1. 3. Then, the sample after 4. 50 Mm ammonium the reductive bicarbonate solution alkylation in 0.5 mL was placed in Column 96-well plate was 6 (A6) of the 12- transferred into the channel tank at PVDF filter plate to Position 7. perform centrifugation. 4. The robotic arm transferred 100 μL 50 mM ammonium bicarbonate solution to wash the sample, and the mixture is then performed with centrifugation. Protein 1.1 The robotic arm 1.1 Trypsin and 1.1 Trypsin 1.1 1 min digestion transferred 100 μL of 50 lysinase (LysC) were and lysinase 1.2 1 min mM ammonium placed in Column 1 of (LysC) 2. 2 h bicarbonate solution and the low temperature 3. 40% 3. 1 min 1 μg of mixed trypsin and disk (namely, the acetonitrile lysinase (LysC). enzyme storage unit) (containing 1.2 The mixture was at Position 6. 0.1% formic added into the PVDF 1.2 The PVDF filter acid) solvent filter plate respectively. plate was placed at 2. Then, the PVDF filter Position 9. plate was displaced to a 2. The thermostatic thermostatic mixing mixing shaker was shaker for shaking placed at Position 1. incubation at 37° C. at a 3. A solvent of 40% rotation speed of 1000 acetonitrile (containing rpm for 2 h. 0.1% formic acid) was 3. After the completion of placed in Column 7 the incubation, the (A7) of the 12-channel peptide fragment filtrate tank (namely, the was collected by treating fluid supply centrifugation. The unit) at Position 7. robotic arm transferred 150 μL of 40% acetonitrile (containing 0.1% formic acid) and added the same to the PVDF filter plate for centrifugal operation. Finally, all the peptide fragment filtrates were combined. Concentrating 1. The combined filtrates 1. The 0.5 ml 96-well 2.1 Aqueous 1. 1 min and were concentrated and plate sample disk was solution 2.1 1 min lyophilizing lyophilized in a vacuum placed on the containing 2.2 1 min centrifugal concentrator. thermostatic mixing 0.1% formic 3. 1 min The peptide fragment shaker at Position 1. acid sample was placed on a 2.1 The aqueous thermostatic mixing solution containing shaker at Position 1 of 0.1% formic acid was the workstation; placed in Column 8 2.1 20 μL of aqueous (A8) of a twelve- solution containing 0.1% channel tank (namely, formic acid was sucked the treating fluid from Column 8 (A8) of supply unit) at Position the twelve-channel tank 7. at Position 7, and added 2.2 The 0.5 mL 96-well into the sample at plate sample disk was Position 1. placed on the 2.2 The mixture was thermostatic mixing vortexed shaker at Position 1 for homogeneously at 1000 vortex mixing; rpm for 1 min to perform 3. The peptide peptide reconstitution. fragment sample after 3. 15 μL of supernatant reconstitution was was transferred from the transferred to the PCR 0.5 mL 96-well plate at plate at Position 3. Position 1 into the PCR plate at Position 3 respectively.

[0106] The chromatographic and mass spectrometric detection parameters in this embodiment are as follows.

[0107] On-line detection of liquid phase parameters: a mobile phase A is set as an aqueous solution containing 0.1% formic acid and a mobile phase B as 80% acetonitrile containing 0.1% formic acid, with gradient elution conditions as shown in Table 2. The chromatographic column is Acclaim™ PepMap™ 100 C.sub.18 (Thermo Fisher, 0.075 mm, 20 mm), with the column temperature of 55° C.

TABLE-US-00002 TABLE 2 Gradient Elution Table Time Mobile Phase Mobile Phase Flow Rate (min) A B (nL/min) 0 99 1 300 1 99 1 300 3 94 6 300 6 92 8 300 23 70 30 300 27 1 99 300 30 1 99 300

[0108] On-line detection of mass spectrometry parameters. A mass spectrum full scan resolution is 60,000@m/z 200. AGC is 3E6. The maximum ion injection time is 100 ms. The scan range is m/z 200-2000. The normalized collision energy is 27%. The secondary mass spectrum scan resolution is 15,000@m/z 200. The scan range is m/z 200-2000. AGC is 1E6. The maximum ion injection time is 50 ms. The dynamic exclusion time is 40 s. The charge valence state is 2.sup.+-8.sup.+.

[0109] After the completion of sample detection, the quantitative intensity of all samples was statistically analyzed (FIG. 4). The results showed that the quantitative results of all samples spanned 6 orders of magnitude. The peptide fragment intensity may be detected from the lower 4 to the higher 10 of intensity (Log 10), and the intensity distribution was stable, indicating that the data coverage was wide and may be used for later analysis.

[0110] As shown in FIG. 5, most of the identified proteins in the samples were distributed between 1000 and 2500, and the number of identified proteins was relatively stable.

Experimental Example 1

[0111] The A, B, C, D samples in Embodiments 1-2 and Comparative Examples 1-2 were all reconstituted with an aqueous solution containing 0.1% formic to the similar concentration of 1 μg/μL for peptide fragment detection by the mass spectrometric detection analysis.

[0112] On-line detection of liquid phase parameters: the mobile phase A is the aqueous solution containing 0.1% formic acid and the mobile phase B is 80% acetonitrile containing 0.1% formic acid. The gradient elution conditions are as shown in Table 2. The chromatographic column is Acclaim™ PepMap™ 100 C.sub.18 (Thermo Fisher, 0.075 mm, 20 mm), with the column temperature of 55° C. On-line detection of mass spectrometry parameters. A mass spectrum full scan resolution is 60,000@m/z 200. AGC is 3E6. The maximum ion injection time is 100 ms. The scan range is m/z 200-2000. The normalized collision energy is 27%. The secondary mass spectrum scan resolution is 15,000@m/z 200. The scan range is m/z 200-2000. AGC is 1E6. The maximum ion injection time is 50 ms. The dynamic exclusion time is 40 s. The charge valence state is 2.sup.+-8.sup.+.

[0113] As shown in FIG. 6, compared with a sample D (the urea method in combination with the traditional pretreatment operation process), the mass spectrum of sample A (the urea final concentration: 6M) has apparently no peptide fragment to be detected. The mass spectrum of sample B (the urea concentration: 4M) has slightly more peptide fragment information than that of the sample A. A sample C (the urea concentration 3 M) has more peaks to be detected, and the detection tendency is consistent with the traditional pretreatment method (sample D). The spectrum is subjected to library search, and the detected peptide fragment information is subjected to protein identification. The statistical results of identification are shown in FIG. 6. Therefore, the PVDF filter plate combined with the urea with the final concentration of 3M is used for pre-proteomic treatment of urine samples, which not only overcomes the defects of traditional FASP method that could not be used for high-flux sample treatment during the protein sample pretreatment, but also improves the detection results of protein in the urine samples.

Experimental Example 2

[0114] This experimental example demonstrates the stability of samples obtained by the urine sample preservation method provided by the invention.

[0115] After the preservation was expired, dithiothreitol was added to the samples in 0 month (i.e., performing subsequent pretreatment operation immediately after protein lysis), 1 month (i.e., after the completion of protein enrichment in Example 3 and the preservation of urine protein in the PVDF filter plate for 1 month), 3 months (i.e., after the completion of protein enrichment in Example 4 and the preservation of urine protein in the PVDF filter plate for 3 months), 5 months (i.e., after the completion of protein enrichment in Example 5 and the preservation of urine protein in the PVDF filter plate for 5 months), 7 months (i.e., after the completion of protein enrichment in Example 6 and the preservation of urine protein in the PVDF filter plate for 7 months), 9 months (i.e., after the completion of protein enrichment in Example 7 and the preservation of urine protein in the PVDF filter plate for 9 months) and 12 months (i.e., after the completion of protein enrichment in Example 8 and the preservation of urine protein in the PVDF filter plate for 12 months), respectively, for a final concentration of 10 mM, and the reaction thereof was carried out at room temperature for 20 min. Iodoacetamide was added to a final concentration of 20 mM and reacted for 20 min in the dark. An equal amount of dithiothreitol was added to neutralize the excess iodoacetamide. 200 μl of 70% ethanol was added and the mixture was centrifuged at 1000 g for PVDF filter plate activation. 200 μL 3M urea (diluent: 50 mM ammonium bicarbonate) was added. The mixture was centrifuged at 1000 g for PVDF filter plate equilibration. The sample was then transferred to the PVDF filter plate and centrifuged at 1000 g. Finally, 50 mM ammonium bicarbonate solution was added to wash the sample, and the mixture was centrifuged at 1000 g. 100 μL of 50 mM ammonium bicarbonate solution and 1 μg of mixed trypsin and lysinase (LysC) were added. The mixture was incubated at 37° C. with shaking for 2 h. After the completion of incubation, the mixture was centrifuged at 1000 g for 1 min for collecting the filtrate. Then 150 μL of 40% acetonitrile (containing 0.1% formic acid) was added to elute the peptide fragments. The filtrates were combined, concentrated and lyophilized, redissolved to 1 μg/μL with aqueous solution containing 0.1% formic acid, and analyzed by the mass spectrometry for peptide fragment detection.

[0116] On-line detection of liquid phase parameters: the mobile phase A is set to the aqueous solution containing 0.1% formic acid and the mobile phase B to 80% acetonitrile containing 0.1% formic acid. The gradient elution program is shown in Table 2. The chromatographic column is Acclaim™ PepMap™ 100 C.sub.18 (Thermo Fisher, 0.075 mm, 20 mm), with the column temperature of 55° C.

[0117] On-line detection of mass spectrometry parameters. A mass spectrum full scan resolution is 60,000@m/z 200. AGC is 3E6. The maximum ion injection time is 100 ms. The scan range is m/z 200-2000. The normalized collision energy is 27%. The secondary mass spectrum scan resolution is 15,000@m/z 200. The scan range is m/z 200-2000. AGC is 1E6. The maximum ion injection time is 50 ms. The dynamic exclusion time is 40 s. The charge valence state is 2.sup.+-8.sup.+.

[0118] The statistical results of protein detection in this experiment are shown in FIG. 7. From the statistical results of protein detection, it is found that when the urine protein is stored in the PVDF filter plate for 12 months, the number of protein and peptide fragments detected on line is relatively stable, and is maintained within the range of 1500 protein and 12000 peptide fragments, with no significant fluctuation. Therefore, the invention provides a preservation method for an urine sample which is reliable and can ensure stable protein quantity and quality within 1 year.

[0119] The above mentioned are merely preferred embodiments of the invention and not intended to limit the invention. There are various modifications and changes in this invention for those skilled in the art. Any modifications, equivalents, improvements, etc. within the spirit and principles of this invention are intended to be included within the scope of this invention.